While the vast majority of individuals are susceptible to HIV infection, a rare subset remains uninfected even after multiple exposures. Research on these persons has led to the identification of a genetic polymorphism (CCR5 32) that, in the homozygous state, provides protection against acquisition. However, this finding accounts for only a minority of persons who remain uninfected after exposure; therefore, other mechanisms must be acting to confer protection. In this application we propose an exploratory analysis of genetic and innate host defense factors that may play a role in protection from HIV infection. With this application we propose to examine mechanisms of protection among cohorts of a) 200 highly- exposed seronegative (HESN) men who have sex with men (MSM), b) 60 low risk MSM controls (LRCs), and c) 60 HIV infected men. In a departure from previous initiatives and a markedly innovative aspect of our proposal, we will not only select subjects with an extremely high self-reported risk of HIV exposure, but will attempt to delineate in exquisite detail those wih definitive evidence of biological exposure by using a multi- layered approach to detect HIV-specific anamnestic responses in the peripheral circulation and in the mucosa. With a robust discrimination of subjects most likely to have been exposed, we will examine intrinsic resistance to HIV replication at mucosal sites and in peripheral blood. We will examine the expression and occupancy of the HIV coreceptor CCR5 by peptide inhibitors at mucosal sites of HIV exposure and will examine the quiescence of mucosal T cells as well as in circulating T cells that we firs reported in HESN hemophiliacs. We will explore in fine detail the role of innate defenses provided by natural killer (NK) and dendritic cell (DC) populations in mucosal and systemic protection from HIV infection., Among subjects who are not CCR5 32 homozygous we will examine the genetic determinants of protection by whole-genome sequencing of blood samples from 250 HESN (50 HESN will be collected in addition to the 200 used for all other studies to increase power of the genetic analyses). Transcriptional profiles will also be performed on rectal mucosal samples from 150 HESN and 60 low risk MSM controls. Using a set of validated virologic assays, impaired infectibility will be localized to specific stages of the viral life cyce to facilitate precise identification of the source of the defect. Finally, these data sets will be interrogated to explore in greater detail the genetic determinants of functional hits and the functional implications of high probability genetic hits in order to harmonize these different search approaches. These novel and comprehensive analyses combining immunologic, virologic and genetic approaches using cohorts at carefully defined risks of HIV exposure will provide critical information on the determinants of mucosal protection from HIV infection.